This project is concerned with pathogenesis in the dark-pigmented fungus Wangiella dermatitidis, an opportunistic pathogen of humans that is a cause of subcutaneous and systemic infections termed phaeohyphomycoses. The primary aim is to test the hypothesis that melanin synthesis in Wangiella is linked to the generation of high intracellular (turgor) pressures that are necessary for invasive growth. This idea is founded on a well-established relationship between melanin and fungal pathogenicity, and on prior research on the plant pathogen Magnaporthe grisea, that causes rice blast disease. The turgor pressure generated by yeast cells and hyphae of wild-type and melanin-deficient strains of Wangiella will be determined from measurements of the melting point of ice crystals within individual cells. This is a new technique in which the crystallization and melting of intracellular ice are controlled with a cold stage. The effect of pigmentation on cell wall permeability will also be tested and permeability data will be correlated with the turgor measurements to study their association. Spectroscopic methods will be used to examine changes in cellular osmolyte composition related to melanization and any measured turgor buildup, and experiments on invasive growth by melanized and non-melanized strains of Wangiella will provide information of clinical significance. These experimental aims are unified by the hypothetical link between pigmentation and pressure which underpins the whole project. Because Wangiella can be cultured safely using standard laboratory facilities, it is a superb model for student research on fungal pathogenesis, and information gathered from this study may also be applicable to diseases caused by more virulent human pathogens. Therefore, the planned experiments on Wangiella may ultimately suggest new approaches for controlling diseases caused by dematiaceous fungi. This project is designed to fulfill the goals of the Academic Research Enhancement Award (AREA) Program and will therefore enrich both mycological research and teaching at Miami University.